Acetylcholine is a neurotransmitter that plays a role in many aspects of hearing, including selective attention, learning, frequency selectivity, sound localization, and discrimination of speech sounds. It also plays a critical role in helping the bran adapt during normal development, during aging and in response to damage of the ear or central nervous system. The long term goal of this research is to understand how cholinergic inputs to brainstem auditory circuits contribute to these tasks. Recent studies have identified 4 different cholinergic systems that innervate the brainstem auditory pathways. These cholinergic systems have different functions such as arousal, setting neuronal sensitivity, or controlling the flow of auditory information (e.g., to determine whether an acoustic stimulus is consciously perceived). A major obstacle to understand cholinergic functions in the brainstem has been lack of information about which cholinergic sources contact which auditory pathways. The objective of this proposal is to identify brainstem auditory circuits that are major targets of specific brainstm cholinergic projection systems. The experiments will use recently developed viral vectors and genetically-engineered (transgenic) rats to label cholinergic circuits from identified sources. These techniques will be combined with multi-labeling anatomical tracers and immunochemistry to identify the components and synaptic organization of specific auditory circuits targeted by the cholinergic systems. The Aims focus on two brainstem areas that show high levels of cholinergic innervation: the ventral cochlear nucleus (VCN) and the inferior colliculus (IC). Together, these 2 areas process nearly all auditory information and thus contribute to all aspects of auditory function. Aim 1 will focus on the VCN, a region that receives direct input from the ear and that gives rise to multiple pathways to higher centers. The experiments will identify cholinergic inputs to specific ascending pathways. Aims 2 and 3 will focus on the IC, the largest brainstem auditory center and a major hub for integration of auditory information. Aim 2 will identify specific cell types in the IC that are targeted by cholinergic inputs. Aim 3 will idetify the circuits within the IC that are likely to be modulated by the cholinergic inputs and that could control how auditory information is processed within this integrative center. Overall, results from the three Aims will move the field forward by providing essential information for designing and interpreting future experiments with optogenetics, physiology and behavior to better understand cholinergic roles in normal hearing, during development, learning and aging and after damage to the cochlea or central auditory system.